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A timeline of milestones in the development of oncolytic virotherapy to improve virus specificity, potency, delivery and spread.
Figure 1: A timeline of milestones in the development of oncolytic virotherapy to improve virus specificity, potency, delivery and spread. Center for Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada.
Oncolytic viruses have the potential to complement many of today's standard cancer therapies. HSV has many attractive features as a viral vector; the genome is very large and can accommodate large inserts, it efficiently infects most cell types both dividing and non-dividing from a broad range of species, it naturally undergoes a latent infection in neurons that causes no detectable damage to the infected cell or can undergo a lytic infection that is cytotoxic, and antiviral drugs are available to treat adverse events. We are continuing to construct new oncolytic HSV vectors and explore the consequences of different mutations on HSV biology in cancer cells. In this strategy, viral replication and associated cytotoxicity are restricted to a specific cell type by the regulated expression of an essential immediate-early viral gene product (ICP4). As many cancer patients are already receiving a variety of anticancer drugs, we have been examining the interactions between oncolytic HSV vectors and these drugs and novel small-molecule inhibitors. In addition to cancer cells, the tumor microenvironment is composed of an array of cell types including: endothelial cells of the neovasculature, inflammatory cells and fibroblasts in the stroma. The host immune response plays a complex role in therapeutic efficacy, for example acute innate responses can inhibit virus spread, whereas, activation of dendritic cells and antigen cross-presentation can prime anti-tumor adaptive immune responses, with oncolytic HSV acting as an "in situ vaccine." We are studying the interaction of HSV with dendritic cells and exploiting this for therapy (related research article). Cancer stem or tumor-initiating cells have recently been described for a number of solid tumors, including glioblastoma.
We have had a long-standing interest in the use of oncolytic HSV to treat prostate cancer, and have used a wide variety of tumors models, including: transgenic mice (related research article), lung metastases (related research article), and primary organotypic cultures (related research article). Neurofibromatosis (Nf) types 1 and 2 are inherited cancer syndromes causing tumors of the peripheral nervous system. Oncolytic Newcastle disease virus (NDV) might be a promising new therapeutic agent for the treatment of pancreatic cancer.
Replication kinetics of rNDVs in 6 different human pancreatic adenocarcinoma cell lines (HPACs). Compared to rNDV-F0, expression of NS1 from rNDV-NS1-F0 did not improve the oncolytic effect for most cells. Abstract Targeted therapy of cancer using oncolytic viruses has generated much interest over the past few years in the light of the limited efficacy and side effects of standard cancer therapeutics for advanced disease. Creative Commons Attribution License (CC BY) which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Imlygic, a cancer drug developed by biotech company Amgen, uses a genetically modified herpes virus that’s programmed to kill tumor cells in patients with melanoma that recurs after surgery.
According to the FDA, 16.3% of participants in a clinical study saw a decrease in their lesions that lasted for at least 6 months.
However, most, if not all current oncolytic vectors are confronted with resistance phenomena severely limiting the efficiency of virotherapeutic approaches. Expression of viral proteins will eventually lead to immune-mediated lysis of infected cells by CD8+ T cells which recognize viral peptide epitopes presented by major histocompatibility complex class I molecules on the surface of the infected cell. Our major focus is the application of oncolytic (replication-competent) HSV for the treatment of cancer.
Because of the interaction of some HSV genes with cellular survival pathways, mutations in these genes can sensitize infected cells to drugs targeting these pathways (related research article). The tumor microenvironment is critical to tumor progression and "armed" oncolytic HSV vectors are being developed that combine direct cell killing with expression of transgenes to inhibit tumor stroma and vasculature (related research article). We have been isolating and characterizing glioblastoma stem cells (GSCs) from patient tumor specimens (related research article). Oncolytic HSV expressing IL12 is particularly effective for prostate cancer, enhancing anti-tumor immune responses and anti-angiogenesis. They are relatively benign tumors except for malignant peripheral nerve sheath tumors (MPNST), which arise in a small proportion of NF1 patients and are usually lethal.
IntroductionPatients with pancreatic adenocarcinoma still have very poor survival rates, and current therapies are of limited effect [1,2]. Modulation of IFN Response by rNDVsAs IFN has a direct effect on oncolyis, the capacity of the viruses to induce, enhance or block IFN production was evaluated. Analysis of Host Immune Response in GLV-1h68-Infected and Noninfected Primary TumorsIn order to analyze the effects of virus infection in vivo, we determined the mouse antigen profiling of GLV-1h68-infected and noninfected tumors of MTH52c or ZMTH3 xenografted nude mice.
Retinoblastoma protein (pRb) is normally hypophosphorylated and binds to transcription factors of the E2F family to regulate the G1-to-S checkpoint of the cell cycle. After intravenous injection, viruses are neutralized by pre-existing antibodies and complement activation. In 2006, the world witnessed the first government-approved oncolytic virus for the treatment of head and neck cancer.
That’s much less than other new cancer treatments, which typically cost more than $100,000, with some new treatments costing more than $250,000 in the first full year of treatment. The aim of the project is to unravel and understand basic mechanisms of resistance to oncolysis and to define novel strategies helping to overcome the current limitations.
Alternatively, cells might be lysed (i) owing to an overwhelming amount of budding and release of progeny virions from the cell surface, (ii) by activation of apoptosis during the course of viral replication and gene expression, or (iii) by massive syncytia formation being directly exerted by viral fusogenic membrane glycoproteins (TCR, T cell receptor). Tumor selectivity is achieved by mutating the virus so that it is permissive in cancer cells, but not "normal" cells, thereby targeting tumors for destruction while being non-pathogenic. A technical advance has been the generation of HSV-BAC (bacterial artificial chromosome) plasmids that can be manipulated in bacteria and biochemically (related research article).
GSCs are very efficient at forming tumors in the brain in immune-deficient mice and their xenografts histologically recapitulate the features present in the corresponding patient tumor. Expression of additional proteins did not hamper virus replication or cytotoxic effects on itself. Oncolytic Newcastle disease virus (NDV) could be a promising new therapeutic agent for the treatment of pancreatic cancer. The nucleotide length of the NDV genome is noted below the plasmid names and all full-length plasmids and pGEM-T-PM-cassette are drawn to scale. 0.1 and samples were taken at indicated time points and titrated by end-point dilution assay in Vero-118 cells. Ten HPACs, as well as Vero cells (lacking the endogenous IFN genes) and MRC-5 cells (fully IFN competent), were inoculated with the five different rNDVs and at 24 h after inoculation both hIFN? protein production as well as gene expression levels were measured. Graphs with specific xenograft-treatment combinations depict individual mouse tumor volumes with black lines and dots. The single vaccinia virus injection caused an efficient inhibition of tumor growth in all GLV-1h68-treated tumor-bearing mice compared to control mice (Figure 6(a)). Canine mammary ZMTH3 adenoma tumors are susceptible to GLV-1h68 treatment in vivo [12] and therefore were used as an additional control. Upon stimulation by mitogenic signals, upregulation of cyclins enables cyclin-dependent kinases (CDKs) to phosphorylate pRb, releasing E2F that leads to the expression proteins needed for DNA synthesis and thus cell cycle progression. It has been known for many years that viruses have the ability to replicate in and lyse cancer cells. Amgen said in a news release that it expects the drug to become available starting next week. That means it won’t infect the person with herpes, but instead attack the cancerous cells directly, and may stimulate an immune system response to tumors especially in combination with other chemotherapy drugs.


Furthermore, concepts of multimodal therapies newly introducing oncolytic viruses as means to overcome resistance to clinically established treatment approaches are developed and evaluated in a preclinical setting. These constructs are useful for rapidly inserting a variety of therapeutic transgenes into defined sites in the HSV genome and to generate transcriptionally-targeted vectors. Thus they provide an excellent model for the development of novel therapeutics and oncolytic HSV against glioblastoma. However, expression of interferon, but not NS1, resulted in loss of multicycle replication. Newcastle disease virus (NDV) has been described as a naturally occurring oncolytic virus as early as 1952 [3,4,5].
The 3?-terminal leader, intergenic regions and 5?-terminal trailer are shown as horizontal lines. The gene expression assay for endogenously expressed IFN maps to the extreme 3? end of the hIFN? gene, with the reverse primer annealing downstream of the stop codon of the hIFN? coding sequence. Last observed tumor volumes were carried forward to calculate median volumes per group in the lowest plots.
Upon necropsy of these animals, only very small residual tumors were found, as opposed to the mostly large tumors in animals treated with either PBS or lentogenic rNDVs. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.AbstractCanine mammary carcinoma is a highly metastatic tumor that is poorly responsive to available treatment. At 42 days after virus injection, MTH52c and ZMTH3 tumors of nude mice with or without GLV-1h68 treatment were removed and used for generation of tumor tissue lysates as described in Material and Methods. On binding to viral double-stranded RNA (dsRNA), PKR autophosphorylates, which in turn phosphorylates the ? subunit of eIF-2. Recent work has revealed that Ad5 binds to erythrocytes via the Coxsackie and adenovirus receptor (CAR) and complement receptor 1 (CR1) in the absence and presence of anti-Ad5 antibodies, respectively[178]. Although encouraging results have been demonstrated in vitro and in animal models, most oncolytic viruses have failed to impress in the clinical setting.
It will be used to treat lesions, or the areas of skin that still have moles and other marks related to melanoma, if they pop up again after being surgically removed. Trial data indicate a 26% complete response (8 of 50), with durability in both injected and uninjected lesions including visceral sites19.
Our general approach is to identify and mutate suitable viral genes, characterize the oncolytic HSV vectors in vitro, test their efficacy and mechanism of action in tumor models in vivo, confirm their safety, and finally translate to the clinic. Conversely, increasing the virulence (rNDV-F3aa) resulted in enhanced replication of the virus. Since then, numerous clinical trials have employed wild type NDV strains either as a direct oncolytic agent, or as an oncolysate vaccine for treatment of patients with various types of advanced stage cancer [6,7,8,9,10,11]. Gene-end and gene-start sequences in the PM intergenic region are depicted as vertical black and white rectangles, respectively.
Results are presented as fold change gene induction of treated versus mock treated cells calculated using the 2???CT method [31]. Sequestration into other organs and the reticuloendothelial system is a particular problem, often with resulting toxicities. The explanation is multifactorial, determined by the complex interactions between the tumor and its microenvironment, the virus, and the host immune response. We utilize a variety of animal tumor models; human xenografts in immune-deficient mice, syngeneic mouse tumor implants, or spontaneously arising tumors in transgenic mice. Results of these early trials have been relatively disappointing, illustrated by the lack of further development of these treatment strategies. Means and ranges of triplicate experiments are plotted; (b) Supernatants were tested for functional IFN protein content using an ISRE-luc bioassay [14]. Recently, we reported that the oncolytic vaccinia virus GLV-1h68 could be a useful tool for therapy of canine mammary adenoma in vivo. From the blood stream, viruses have to pass through a mixture of extracellular matrix, cells (including normal and immune cells) and high interstitial fluid pressure before reaching the tumor.
This review focuses on discussion of the obstacles that oncolytic virotherapy faces and recent advances made to overcome them, with particular reference to adenoviruses. Intravenous delivery of JX-594, an oncolytic vaccinia virus, in patients with metastatic tumors has demonstrated the need for a viremic threshold to be reached for efficient virus delivery to tumors29 (2011). With the advent of recombinant DNA techniques it has become possible to genetically engineer NDV [12], and interest in the use of recombinant NDV (rNDV) as an oncolytic virus has revived over the last decade [13]. RNA of indicated rNDV stocks was reverse transcribed into cDNA and the PM intergenic region was amplified with RT-PCR using flanking primers; (c) Western blot for expression of NS1 protein.
These findings were mostly in agreement with our findings using wild type NDV, except for BxPC-3 cells [14]. In this study we analyzed the therapeutic effect of GLV-1h68 against canine mammary carcinoma. Two-way analysis of variance (ANOVA) was used to compare the two corresponding data points of the two groups.
They then have to attach to the cellular receptor (often trapped in tight junction), be internalized, translocate to the nucleus, replicate, produce structural and other proteins, lyse the cell and release their progenies – some of these steps could be inhibited by factors such as the natural host immune response, hypoxic environment, soluble factors, and genetic changes in the tumor cell.
Evaluation in vivo using mice bearing subcutaneous pancreatic cancer xenografts revealed that only intratumoral injection with rNDV-F3aa resulted in regression of tumors. NDV selectively replicates in and destroys tumor cells while sparing normal cells, presumably because of defective interferon signaling pathways of the innate immune system in tumor cells. Whole cell lysates of Vero-118 cells (mock-) inoculated with indicated viruses were assayed for expression of the NS1 protein or tubulin protein. Cell culture data demonstrated that GLV-1h68 efficiently infected and destroyed cells of the mammary carcinoma cell line MTH52c.
Inactivated IFN and activated Ras pathways are frequently found in cancer (the latter could inhibit PKR), and some naturally-found viruses can replicate selectively in cancer but not normal cells, including the Newcastle disease virus (NDV)[21], reovirus[22], vaccinia virus[23], and vesicular stomatitis virus (VSV)[24].
We conclude that although lentogenic rNDVs harboring proteins that modulate the type I interferon pathway proteins do have an oncolytic effect, a more virulent mesogenic rNDV might be needed to improve oncolytic efficacy. Previously, we reported that a lentogenic wild type NDV strain replicated in and was cytotoxic for 11 human pancreatic adenocarcinoma cell lines (HPACs) with high variability. Furthermore, after systemic administration, this attenuated vaccinia virus strain primarily replicated in canine tumor xenografts in nude mice. Hellmen, “Complex mammary tumours in the female dog: a review,” Journal of Dairy Research, vol.
The herpes simplex virus (HSV) protein ICP34.5 interacts with cellular phosphatase 1? to dephosphorylate eIF-2?, leading to synthesis of proteins needed for virus replication. These differences in the response of HPACs were not due to defects in innate immunity pathways as a number of these cell lines produced type I interferon (IFN) upon NDV infection [14]. Finally, infection with GLV-1h68 led to strong inflammatory and oncolytic effects resulting in significant growth inhibition of the tumors. We, and others, have also shown that IFN produced by tumor cells and the normal cells surrounding the tumor cells exerts anti-proliferative, pro-apoptotic and pro-inflammatory effects in tumor cells, possibly attributing to oncolytic efficacy [15,16,17,18,19]. In summary, the data showed that the GLV-1h68 virus strain has promising potential for effective treatment of canine mammary carcinoma.1.
Because cancer cells are often in the S phase, E1A CR2-deleted Ad5 mutant (dl922-947) could selectively replicate in and destroy replicating cancer cells but not normal resting cells[29].
Therefore, rNDVs armed with the IFN gene might result in a virus with higher oncolytic efficacy.


To test whether the differences in expression of the hIFN? genes also resulted in differences in protein production, functional IFN protein content was determined in the supernatants of inoculated cells. IntroductionMalignant tumors of the mammary glands are among the most frequently observed tumors in female dogs [1–3]. Due to biosafety issues, we could not measure IFN in supernatants of cells inoculated with rNDV-F3aa.
Despite the success in diagnosis and treatment of mammary cancer, this disease entity remains one of the leading causes of cancer-related death in female dogs.
Sorenmo, “Canine mammary gland tumors,” Veterinary Clinics of North America—Small Animal Practice, vol. As such, engineered VAI-deleted adenovirus (dl331) could replicate selectively in tumors with an activated Ras pathway[27].
The tumor selectivity of E1B 19K-deleted Ad2 (dl250) is due to multiple defects in the apoptotic pathways, where survival of the virus in normal cells would be limited owing to rapid apoptosis induction in the presence of tumor necrosis factor-? (TNF-?)[30]. Viruses use different strategies to counteract the IFN pathway to increase infectivity and replication efficiency. Upon inoculation with rNDV-F0, cells demonstrated variation in the extent of IFN production. Therefore, there is an urgent need to identify novel agents for therapy and diagnosis of mammary cancer.
Epstein-Barr virus (EBV) also expresses RNAs similar to VA RNAs and these can complement dl331, resulting in selectivity in EBV-associated tumors[28]. E1B 55K interacts with the adenovirus E4 open reading frame 6 (E4orf6) protein to form an E3 ubiquitin ligase complex that targets p53 for degradation.
The non-structural NS1 protein of influenza A virus is one of the most potent antagonists of the IFN response of the innate immunity characterized to date [20,21].
Six of the 10 HPACs produced significant less IFN compared to IFN-competent MRC-5 cells, while HPAF-II, CFPAC, and Hs 766T had a similar range of IFN production.
Among the most promising new therapeutic candidates are oncolytic viruses, which can target tumor tissue and specifically eradicate the cancer cells.
It also induces the expression of cyclin E as well as simultaneously inhibits cellular mRNA export and promotes the export of late viral mRNAs. Oncolyis of tumor cells depend on efficient virus infection and replication, therefore, incorporation of an IFN antagonist, such as the NS1 protein of Influenza A in the genome of rNDV, could be a way to improve the oncolytic efficacy of rNDV. This concept was already confirmed in human tumor xenograft treatment by the use of several viruses [4–9].
NDV strains are categorized in three different groups based on the severity of the disease they cause in birds: lentogenic, mesogenic, and velogenic and this classification correlates with their oncolytic properties in cancer cells. In the present study, we tested the recombinant oncolytic vaccinia virus GLV-1h68 as a therapeutic agent against canine mammary carcinoma. Increasing the virulence of rNDV has been shown to improve direct oncolytic efficacy most, resulting in preclinical studies using mesogenic virulent rNDVs expressing transgenes, such as IFN and the NS1 protein of influenza virus [13].
However, virulent NDV strains pose an environmental risk, as birds (specifically poultry) are very susceptible to infection with mesogenic or velogenic strains.We hypothesize that arming lentogenic rNDV with IFN-modulating genes, either accelerating or blocking the IFN response, would improve the oncolytic effect of NDV sufficiently to circumvent using virulent rNDV with the associated potential biosafety risks. Inoculation of HPACs and MRC-5 cells with rNDV-NS1-F0 resulted in a significant decrease of IFN production in all these cells as compared to cells inoculated with rNDV-F0 (Figure 3b). Recombinant Virus RescueRecombinant NDVs were rescued using a method adapted from the original method described previously [12]. Three days later, 200 µL BSR-T7 supernatant was injected into the allantoic cavity of 10 day old specified pathogen free (SPF) embryonated chicken eggs.
Kirn, “From ONYX-015 to armed vaccinia viruses: the education and evolution of oncolytic virus development,” Current Cancer Drug Targets, vol.
After incubation in a humidified egg incubator at 37 °C for two or three days (mesogenic or lentogenic rNDVs, respectively), allantoic fluid was harvested and presence of virus demonstrated by hemagglutination assay [25]. Samples displaying hemagglutination were passaged once more in eggs to increase virus titer, and allantoic fluid was harvested after 2 or 3 days. Kirn, “Clinical trial results with oncolytic virotherapy: a century of promise, a decade of progress,” Nature Clinical Practice Oncology, vol. Characterization of rNDVRNA from virus stocks was isolated using the High Pure RNA Isolation kit (Roche, Woerden, The Netherlands) following the manufacturer’s instructions and reverse transcribed into cDNA using a two-step protocol, as described before [14]. Whole cell-lysates taken 18 h post-inoculation were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a Hybond-C Extra nitrocellulose membrane (GE Healthcare Life Sciences, Diegem, Belgium).
Chemiluminescent signals were generated using Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare Life Sciences) following manufacturer’s instructions and detected using a ChemiDoc MP system (Bio-Rad, Veenendaal, The Netherlands). Barry, “Reprogrammed viruses as cancer therapeutics: targeted, armed and shielded,” Nature Reviews Microbiology, vol. 0.1, in triplicate After a 1 h incubation, cells were washed three times with PBS and fresh medium was added.
Samples were titrated by end point dilution assay in quadruplicate as described before [14]. Thorne, “Targeted and armed oncolytic poxviruses: a novel multi-mechanistic therapeutic class for cancer,” Nature Reviews Cancer, vol. 3, cells were lysed with 300 µL lysis buffer of the Total Nucleic Acid Isolation kit (Roche) and RNA was isolated using a MagNA Pure LC machine (Roche) following the manufacturer’s instructions. To detect both endogenous and exogenous expressed IFN, primers and probes mapping in the IFN coding region were used with an in-house developed assay and ?-actin was used as household gene). The sequences of the primers and probes for the IFN-coding region and ?-actin have been described before [30].
Results are presented as fold change of inoculated samples versus mock-inoculated samples (duplicates), calculated using the 2???CT method [31]. Szalay, “The immunologic aspects of poxvirus oncolytic therapy,” Cancer Immunology, Immunotherapy, vol. At time point 120 h post inoculation, cell viability was determined using the CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, Leiden, The Netherlands), as described before [14].
Prism for Windows version 5.03 (GraphPad software, La Jolla, CA, USA) was used to analyze data using the log(inhibitor) vs. Wang et al., “Eradication of solid human breast tumors in nude mice with an intravenously injected light-emitting oncolytic vaccinia virus,” Cancer Research, vol.
The experimental protocol was reviewed and approved by an independent animal experimentation ethical review committee, not affiliated with Erasmus MC (DEC consult number EMC2921). Brader et al., “Novel oncolytic agent GLV-1h68 is effective against malignant pleural mesothelioma,” Human Gene Therapy, vol. Amplicons of the P-M intergenic (Figure 1b) and F protein cleavage site were sequenced to confirm identity of the rNDV stocks. Upon titration of virus stocks, only infection with rNDV-F3aa lead to syncytia formation in cell cultures, characteristic for infection with mesogenic NDV (data not shown). In addition, Western blot assay confirmed the expression of NS1 by rNDV-NS1-F0 (Figure 1c).



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